The Study Of The Role Of GSK-3β In Dexamethasone-induced Pancreatic β Cell Apoptosis In Vitro

Objectives The present study used dexamethasone(DEX) as apoptosis inducer to treat rat pancreatic β-cell line INS-1. To explore the effect of Glycogen synthase kinase-3β(GSK-3β) on glucocorticoid-induced β cell apoptosis, which will provide experimental data for unraveling the underlying mechanism of steroid diabetes.Methods 1 We employed rat pancreatic β-cell line INS-1 as experimental targets.Using techniques of MTT, we examined the cell viability. To determine the optimal concentration and time. 2 Using techniques of Trypan Blue、Tunel、flowcytometry, we examined the cell viability and apoptosis of INS-1. 3 Using techniques of immunofluorescence stain, we examined the expression of p-GSK-3β(Ser9) 、 GSK-3βprotein level. 4 Using techniques of Western-blot 、 Real-time PCR, we examined the expression of SOD、i NOS、Nox4、NADPH oxidase(p47phox)、p-GSK-3β(Ser9)、GSK-3β at m RNA and protein level. We examined ROS level and NO release by ROS kit and Griess method,respectively. 5 The influence of Li Cl on β-cell were observed.Results DEX treated INS-1 at concentrations from 0 to 1μM for 1 to 3 days. Along with the increase of DEX dose and duration, the cell viability of INS-1 progressively decreased.Tunel and flowcytometry results proved that 48 h DEX treatment at concentration of 0.1μM induced maximum of apoptosis. The m RNA expression of Nox4、p47phox、i NOS and protein expression of i NOS 、 Nox4 significantly were increased after DEX treatment,along with the increase of ROS and NO levels and decrease of SOD、p-GSK-3β(Ser9),while total GKS-3β expression was not significantly varied. Li Cl treatment could reduce INS-1 apoptosis and decrease ROS and NO levels, as well as inhibited the m RNA expression of Nox4 、 p47 phox 、 i NOS and protein expression of i NOS 、 Nox4 and SOD、p-GSK-3β(Ser9). The differences all reached statistical significance(P<0.05).Conclusions DEX induced rat pancreatic β-cell INS-1 apoptosis by activating GSK-3βand inhibition of GSK-3β activity by Li Cl could reduce DEX-induced apoptosis to acertain extent,of which mechanism was involved with oxidative stress pathways.